The present invention relates, in a general manner, to means for regulating the disinfection of liquids. It relates more particularly to means of regulation using the measurement of enzymatic activities.
The assurance of the quality and safety of animals, such as water intended for consumption, dietary liquid, bathing water, water intended for pharmaceutical or biotechnological preparations, depends directly on the reliability and the sensitivity of the techniques used in measure the number of microorganisms surviving in the said liquid.
The methods currently used for regulating the disinfection of a liquid use the techniques for culturing on agar and/or microscopy techniques.
The techniques for culturing on agar consist in counting the number of bacterial colonies which develop on various standardized nutrient agar media [e.g. French Standard NF T 90-414, Essais des Eaux, Recherche et dxc3xa9nombrement des coliformes et des coliformes thermotolxc3xa9rants (Water analysis, Identification and Enumeration of coliforms and of thermotolerant coliforms)]. These techniques have several disadvantages.
In the first instance, there may be mentioned the fact that they give their results only after 24 hours on average, which delays by as much the possible adjustment of the disinfection method.
The techniques for culturing on agar necessitates, furthermore, the carrying out of series of microbiological cultures for the analysis of each liquid sample. Indeed, all the microbial, and in particular bacterial, families do not develop on the same nutrient medium; thus, it is possible to detect no coliform after culturing on the standardized nutrient medium for the detection of coliforms, but to find a good number of other bacteria after culturing on other nutrient media. The choice of the nutrient agar media therefore determines the quality of the analysis. This choice is all the more delicate since a larger number of colonies has sometimes been observed after culturing on a medium other than the standardized nutrient medium for the detection of these same colonies. It has, for example, been demonstrated that the counts of the viable aerobic bacterial flora were higher on R2A agar medium than on the corresponding standardized nutrient medium (see for example  less than  less than A new rapid medium for enumeration and subculture of bacteria from potable water greater than  greater than  by Reasoner D. J. and Godreich E. F., App. Environm. Microbiol. (1985) 49-p.1-7). To establish a reliable negative diagnosis, the techniques for culturing on agar therefore require a multiplication of the analyses. The techniques for culturing on agar furthermore do not easily allow the regulation of the method of disinfection to be automated.
Bacteria are furthermore capable, and in particular following an environmental stress such as the application of a disinfection method, of adopting a form of resistance in which they no longer multiply while providing a minimal metabolism; as soon as more favourable environmental conditions are restored, these bacteria could resume their multiplication. Such bacteria are said to be  less than  less than non-culturable but viable greater than  greater than : they are not detectable by the conventional techniques for culturing on agar and can represent a biological risk for the consumer.
A method which is currently available for controlling with virtual certainty the efficiency of the disinfection of a liquid uses microscopy techniques combined with specific staining. This method allows discrimination between culturable bacteria, non-culturable but viable bacteria and dead bacteria. It requires nevertheless the use of several staining tests for each sample and is technically difficult to automate.
The present invention aims to overcome the disadvantages of the prior art techniques and proposes a method for regulating the disinfection of a liquid, characterized in that it comprises:
A. at one stage of the said disinfection, designated hereinafter stage 2, measuring the activity of at least one enzyme by bringing the microorganisms which may be present in the said liquid into contact with a substrate chosen as being capable of revealing the activity of this or these enzyme(s), in particular by the conversion of the said substrate to coloured fluorescent or luminescent compounds or by the disappearance of the said substrate, this enzymatic activity being called hereinafter specific activity,
B. at one stage, designated hereinafter stage 1, prior to the said stage 2, measuring the activity of the same enzyme(s) as in A, this activity being designated hereinafter initial activity,
C. translating, for each enzyme, the said specific activity and initial activity, into levels of surviving microorganisms in the said liquid at stage 2 of the said disinfection by means of a reference system preestablished with the aid of a sample of the said liquid collected at the said stage 1 and then exposed to increasing doses of disinfectant(s), as well as
D. adjusting, as a function of the said level of surviving microorganisms, of the nature and/or doses of physical or chemical agent(s) used for the said disinfection.
The expression surviving microorganisms is understood to mean in the present application culturable microorganisms and/or non-culturable but viable microorganisms.
The expression level of surviving microorganisms is understood to mean in the present application the ratio between the concentration of surviving microorganisms in the said liquid at the said stage 2 of a disinfection and the concentration of surviving microorganisms in the same liquid at the said stage 1. This level of surviving microorganisms is preferably expressed as reduction values in the form of negative powers of 10, or as log reduction which corresponds to xe2x88x92log10 (reduction).
The said stage 1 may, for example, correspond to a stage  less than  less than before disinfection greater than  greater than  of the said liquid and the said stage 2 to any stage of the disinfection method (stages  less than  less than after disinfection greater than  greater than  of the said liquid included).
The method according to the invention relates to liquids in which the microorganisms which may be present are subjected to very specific conditions, namely disinfection conditions, which induce notable stress of the cells.
The method for regulating the disinfection of a liquid according to the invention relates particularly to liquids intended to be brought into contact with humans or animals, whether through simple contact, absorption, ingestion, instillation or injection. It applies particularly to liquids intended to be in contact with humans or animals, such as bathing water, water intended for consumption, water intended for pharmaceutical or biotechnological preparations, or a dietary liquid.
The said bringing of the microorganisms which may be present in the said liquid into contact with the said substrate may be carried out by bringing the said liquid or a sample of the said liquid directly into contact with the said substrate, or alternatively by bringing a concentrate of the said microorganisms which may be present, such as a filtrate or a centrifugation pellet of the said liquid or liquid sample, into contact with the said substrate.
Prior to the measurement of the activity of some enzymes such as glucose-6-phosphate dehydrogenase or glutathione reductase, the method according to the invention advantageously comprises the step of subjecting the said liquid, liquid sample or concentration to a lysis treatment, especially by sonication.
Prior to the measurement of the activity of other enzymes such as catalase or superoxide dismutase, the preliminary lysis stage may be avoided: the activity of these enzymes may be measured with the aid of a substrate which diffuses into the microorganisms, such as lucigenin or hydrogen peroxide.
According to an advantageous aspect of the invention, the enzyme(s) whose activity or activities is (are) measured exhibit(s) in the said liquid, liquid sample or concentrate a ratio between the specific activity and the initial activity in close relationship, and with a slope which is significantly different from zero, preferably less than xe2x88x920.2, with the level of surviving microorganisms over at least one zone of values of the said levels of surviving microorganisms. This is for example the case for glucose-6-phosphate dehydrogenase for log reduction values of between 0 and 3 approximately, for glutathion reductase for log reduction values of between 4 and 7 approximately, and for superoxide dismutase for log reduction values of between 3 and 6 approximately.
Other enzymes of interest may in particular form part of the family of dehydrogenases or the family of enzymes involved in the response to oxidative stress.
To determine if one or more enzymes are appropriate for carrying out the method of regulation according to the invention on a given liquid, the method may be carried out as follows:
collection of at least one sample of the said liquid and measurement of the concentration of surviving microorganisms (culturable microorganisms and/or non-culturable but viable microorganisms), in particular by culturing on agar and/or by staining and observations on a microscope,
assay of the activity of each candidate enzyme on this or these liquid sample(s),
exposure of the said liquid sample(s) to various doses of disinfectant(s) under conditions which are moreover equivalent (e.g. temperature and duration of exposure conditions),
measurement of the activity of each candidate enzyme after exposure to various doses of disinfectant(s),
plotting, for each candidate enzyme, of a curve showing the percentage of activity (enzyme activity, measured after exposure to the various doses of disinfectant(s), expressed relative to the corresponding enzymatic activity before exposure) as a function of the measured log reduction values (as defined above),
determination, for each candidate enzyme, of the log reduction zone for which the slope of the said curve is significantly different from zero, preferably less than xe2x88x920.2, this zone constituting the range of response of the candidate enzyme considered on the said liquid,
a candidate enzyme is appropriate for carrying out the method according to the invention on the said liquid if its range of response comprises the log reduction values corresponding to the disinfection objectives aimed at for the liquid considered.
According to a particularly advantageous aspect of the invention, the or at least one of the enzyme(s) whose activity or activities is (are) measured is a glucose-6-phosphate dehydrogenase, a malate dehydrogenase, a glyceraldehyde-3-phosphate dehydrogenase, a catalase, a superoxide dismutase or a glutathion reductase. The said (or at least one of the said) enzymatic activity or activities is (are) then measured by converting a substrate and, where appropriate, in the presence of a cofactor, such as respectively glucose-6-phosphate and cofactor NADP, L-malate and cofactor NAD, glyceraldehyde-3-phosphate and cofactor NAD, lucigenin, hydrogen peroxide, oxidized glutathion and cofactor NADPH.
According to an advantageous embodiment of the invention, the said measurements of specific activity and of initial activity are made with the aid of an apparatus for detecting light intensity such as a spectrophotometer, spectrofluorometer or a luminometer, optionally having several channels for analysis.
According to another advantageous embodiment of the invention, the said translation into level of surviving microorganisms with the aid of the said preestablished reference system comprises for each enzyme, the calculation of the ratio between the specific activity and initial activity, optionally expressed as a percentage.
According to yet another advantageous embodiment of the invention, the said reference system exists in graph form such as one or more curves relating, for each enzyme, the said ratio between the specific activity and the initial activity to values of level of surviving microorganisms such as log reduction values. The said ratio between specific activity and initial activity is also designated by  less than  less than relative activity greater than  greater than  in the present application.
According to one aspect of this other advantageous embodiment of the invention, the said spectrometric measurements may in particular be carried out, for each enzymatic activity measured, at a constant temperature, in particular at 25xc2x0 C., and at a constant wavelength, in particular at a wavelength of between 240 and 550 nm, for example at 340 nm. The said measurements may be recorded continuously or discontinuously over a time interval of about 30 min. In the case where higher measurement sensitivities are sought, luminometry is preferably used.
The disinfection adjusting stage of the method according to the invention may be carried out by regularly monitoring the variation of the level of surviving microorganisms (e.g. expressed as reduction, log reduction or D index) with the aid of the enzymatic indicators presented above, until the set disinfection objective is met.
The disinfection adjusting stage of the method according to the invention may also be carried out by addition of the  less than  less than disinfectant dose greater than  greater than  equivalent corresponding to the difference between the level of surviving microorganisms aimed at (set value) and the level of microorganisms effectively measured on the said liquid, liquid sample or concentrate at a stage of the or after disinfection.
Advantageously, the said equivalent dose of disinfectant(s) is as read on a reference curve representing the level of culturable microorganisms as a function of the dose of disinfectant(s) to which the said liquid is exposed.
The invention gives particularly advantageous results for liquids comprising microorganisms chosen from the group consisting in particular of the genus Escherichia, Alcaligenes, Bacillus, Flavobacterium, Methylobacterium, Pseudomonas, Klebsiella, Enterobacterium, Agrobacterium, Streptococcus, Micrococcus, Salmonella.
The method for regulating the disinfection of a liquid according to the invention can, in a particularly advantageous manner, be easily automated on a method for disinfecting a liquid. Unlike the prior art techniques, the method according to the invention allows a microbiological control which is reliable, rapid and easy to carry out.
Preferably, the said level of surviving microorganisms is expressed as the value of reduction (concentration of surviving microorganisms expressed in terms of the initial concentration of surviving microorganisms, as measured in the said stage 1), of log reduction (xe2x88x92log10 (reduction)) or of D index (=log reduction). The said D index also constitutes an index of the microbiological quality of the liquid considered.
The said method for regulating the disinfection of a liquid according to the invention considers, as surviving microorganisms, those of the microorganisms present which are culturable and/or those of the microorganisms present which are not culturable but which are viable. In the case where it is desired to take into account both the culturable microorganisms and the non-culturable but viable microorganisms, the said reference system then advantageously relates, for each enzyme, the said ratio between specific activity and initial activity to values of level of surviving microorganisms resulting from the addition of the values of level of culturable microorganisms and of the values of level of non-culturable but viable microorganisms, as measured with the aid of conventional techniques.
Among the said chemical or physical agents which are advantageously used for the said disinfection, there may be mentioned chlorine and its derivatives, UV radiation, ozone, H2O2, filtration membranes, temperature, ultrasound or ionizing radiation.
Other characteristics and advantages of the present invention will also emerge through the following embodiments which are given as a guide with no limitation being implied.